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ISOLATION of TOXIGENIC FUNGI from COMMERCIALLY AVAILABLE MEDICINAL PLANT MATERLAL LUXSHMI FERNANDO and KRISHANTHI ABEYWICKRAMA D

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ISOLATION of TOXIGENIC FUNGI from COMMERCIALLY AVAILABLE MEDICINAL PLANT MATERLAL LUXSHMI FERNANDO and KRISHANTHI ABEYWICKRAMA D J. Natn. Sci. Coun. Sri Lanka 1996 24(1): 1-8 ISOLATION OF TOXIGENIC FUNGI FROM COMMERCIALLY AVAILABLE MEDICINAL PLANT MATERLAL LUXSHMI FERNANDO and KRISHANTHI ABEYWICKRAMA Department of Botany, University of Colombo, Colombo 3. (Received: 26 October 1994; accepted: 06 October 1995) Abstract: Sun-dried commercial samples of six medicinal plant species widely used in Sri Lanka for treatment of diseases were used to isolate saprophytic and pathogenic fungi. Twelve species of fungi were isolated from various parts of plants on potato dextrose agar plates after surface disinfection. Aspergillus flavus (yellow-green form), a notorious toxigenic fungus was isolated from all 6 plants. Other potentially toxigenic species present wered. parasiticus, A. ochraceus, A sulphureus, and Fusarium auenaceum. Duration of storage and environmental conditions *~lav " a vital role in the -erowth and formation of mycotoxins by molds onfin plant products. Screening techniques for known mvcotoxins and aualitv" control methods are essential in reducing- the likelihood of contamination of medicinal plant material. Key words: Aspergillus flavus, fungi, Fusarium, medicinal plants, toxigenic fungi. INTRODUCTION Abrus precatorius Linn. (olinda), Alysicarpus vaginalis (Linn.) DC. (aswenna), Asteracantha longifolia Nees. (neera mulliya), Cassia auriculata Linn. (ranawara),Mollugo cerviana Seringe (paipadagam), and Rauvolfia serpentina (Linn.) Kurz (ekaweriya) are valuable medicinal plants widely used in the treatment of a variety of diseases. These plants occur in tropical regions of India, Sri Lanka, Thailand, Philippines, China and also in Africa and North America. Aqueous extract of roots ofAbrus relieves obstinate coughs and sore throats and is used as a snake-bite remedy. The leaves are used in the treatment of conjunctivitis, painful swellings and stomach complaints. In Sri Lanka, seeds are used to cure itch and sores and wounds.due to biting of mammals and also in the treatment of diabetes.l A. vaginalis is used with other drugs for fever, dysentery, diarrhoea, and chronic malarial fevers.' In Sri Lanka, decoctions of the whole plant are used specifically for dissolving stones in the bladder and also as a diuretic and expectorant.' C. auriculata roots are used in decoctions for fever, diabetes, constipation and diseases of the urinary system. While leaves have laxative properties, dried flowers and buds are used as a substitute for tea in the case of diabetic patients. In Africa, bark and seeds of this plant are used for rheumatism, eye diseases and gonorrhoea.l M. cerviana has antiseptic properties and is also used to cure itch and other skin disorders. The extract of the plant is believed to promote the flow of lochial discharge in w0men.l R. serpentina is used for fever, cholera and to control blood pressure. In India, decoctions of roots are given to increase uterine contractions in chi1dbirth.l 2 L. Fernando and K. Abeywickrama Some of the medicinal plants are restricted to special localities, from where they are collected, dried and then distributedto the rest of the country and stored for future use. Storage decay of plant products is a common occurrence in Sri Lanka. Proper storage facilities are not available in Sri Lanka thus increasing the possibility of medicinal plant contamination by naturally occurring . saprophytic or pathogenic fungi. The levels of relative humidity and environ- mental temperature in tropical countries are precisely the conditions which favour fungal colonization of stored food material. This may result in the accumulation of toxic fungal metabolites, which can be hazardous to humans who consume mold contaminated products without knowing the presence of mycotoxins. Previous reports indicate the occurrence ofA. flavus and Fusarium species on surface disinfected dried Sri Lankan medicinal plant parts ofAerua lanata, A. vaginalis and Tribulus terrestris. 23 These reports further indicate the capability of aflatoxin and Fusarium toxin production by the above mentioned fungi. Sclerotial and non sclerotial isolates ofA. flavus (yellow-green form) from A. lanata and A. vaginalis produced aflatoxin B, levels upto 2800 &50 ml of culture filtrate when grown on a liquid (SMKY) medium and incubated at 25OC for 12 days.2 The present study was conducted to determine the possibility of contamination of some commercially important stored medicinal plants (which are used in decoctions and other ayumedic preparations) with saprophytic, toxigenic or pathogenic mycoflora. METHODS AmlVLATE-s Plant material: Plants ofA. precatorius, A; baginalis,A. longifolia, C. auriculata, M. cerviana and R..serpentina were obtained from randomly selected medicinal plant retail stores in Gabo's Lane, Colombo. These plants had been collected from various localities in Sri Lanka, mixed,. sun dried aild stored in gunny bags at room temperature for 3-4 months. Five 100 g samples of each plant species were purchased from the retail stores. The five samples of each species were mixed prior to analysis. Fungal flora: From each of the above mixed samples, 50 randomly selected pieces of stems, roots, leaves, seeds or flowers were surface sterilized for 5 min in 5% Clorox (0.025% NaOCl) and transferred to Potato Dextrose Agar (PDA) plates containing 0.1 mg& Streptomy~in.~The plates were incubated for 8-10 days at room temperature and the fungi that grew out were identified by comparing their morphology with published books key^.^^ The frequency of occurrence was also calculated. RESULTS A number of fungal species which were previously known for mycotoxin produc- tion were isolated along with few pathogenic or saprophytic mycoflora from surface disinfected plant parts. A yellow-green form of A. flavus, a notorious toxigenic fungus was isolated from all 6 plants used in this study (Table 1). This fungus occurred at highest frequencies on rbots of R. serpentina (84% of total colonies) and A. precatorius (50%). Olive colo~redA. flavus species was isolated Fungi from Medicinal Plants 3 from A. precatorius ,A. vaginalis andA. longifolia. This mold occurred at 8% and 10% frequencies on stems and seeds ofA longifolia respectively. Aspergillus parasiticus, another toxigenic mold, was isolated only from A. longifolia at a frequency of 10%. A. ochraceus and A. sulphureus which have been reported to produce harmful secondary metabolites occurred in roots of A. vaginalis (10%) and A. longifolia (total frequency 24%). Besides potent Aspergillus species, F. avenaceum was isolated at a frequency of 4% from seeds of A. longifolia. Saprophyte A. niger, was isolated from 5 medicinal plants, and occurred at high frequencies on roots of A. precatorius (78%), seeds of A. longifolia (68%) and flowershuds of C. auriculata (64%). Other non-toxigenic fungi present were Neocosmospora vasinfecta, Anthostomella destruens, Curvularia pallescens and Rhizoctonia solani (Tables 1 and 2). Table 1: Fungi present in surface disinfectedplantmaterials ofA.precatorius, A. vaginalis, A. longifolia and their frequencies. Fungi Plant species A. precatorius A. vaginalis A. longifolia 1" 2 1 3 4 A. niger A. flavus (yellow-green) A. flavus (olive) A. parasiticus A. ochraceus A. sulphureus C, pallescens F. avenaceum A. destruens N.vasinfecta Peyronellae spp. R. solani a = plant part: 1= root; 2 = leaves, seeds, and stems; 3 = stems; 4 = seeds; b = percentage colonies, based on colonies present in 50 pieces of plant tissue. DISCUSSION A previous study indicated the occurrence ofsclerotial isolate ofA. jZavus (yellow- green) on surface disinfected leaves, flowers and stems ofAerva lanata and whole plant pieces of A. vaginalis at total frequencies of 35 and 100% re~pectively.~ Another report indicated that out of 50 isolates of A. flauus obtained from different drug plant parts, 21 isolates were toxigenk9 It was reported that the frequency of A. flavus can be as high as 62% in surface disinfected winged bean seeds.1° These results are not surprising since A. fZavus can grow on a wide variety of crops including cereal grains, spices, pulses, vegetables and medicinal plants and also may produce many aflatoxins including aflatoxin B, and B,. A, fZavus therefore may grow readily on medicinal plant parts in tropical countries. lo 4 L. Fernando and K. Abeywickrama Table 2: Fungi present in surface disinfectedplant materials of C. auriculata, M. cerviana, R. serpentina and their frequencies. Fungi Plant species C. auriculata M. cerviana R. serpentina 5" 6 1 A. niger 64b 52 A. flavus (yellow-green) 4 2 84 A. flavus (olive) A. parasiticus A. ochraceus A. sulphureus - C. pallescens F. avenaceum - A. destruens 4 N. vasinfecta Peyronellae spp. 34 R. solani a = plant part: 1 = root; 5 =flower and flower bud; 6 = pieces from whole plant; b= percentage colon~es, based on colonies present in 50 pleces of plant tissue. i' In Sri Lanka, coconut oil and peanut products have been extensively tested and found to contain relatively high amounts of aflatoxins." Processed coconut food products examined by Samarajeewa contained more than 50 pglkg aflatoxin.ll These levels are referred to as medium-high in the Tropical Products Institute classification of toxin levels." Even though not very common and popular as A. flavus, A. parasiticus, A. ochraceus and A. sulphureus have been reported to be toxigenic molds which produce metabolites such as ochratoxin A,B,C, aflatoxins and other derivatives.12In a previous study A. ochraceus and A. parasiticus have beenisolated from surface sterilized Brassica spp. and spices like pepper.13.14This is the first instance where A. sulphureus was isolated from a valuable tropical medicinal plant such as A. longifolia. A toxigenic fungal species, F. avenaceum, which was isolated from A. longifolia seeds has not been recorded in Sri Lanka before. F. avenaceum has a worldwide distribution as a pathogen that causes root, foot and ear rot of cereals and a large number of other hosts. This fungus has been isolated from overwintering cereals in the USSR and is particularly prevalent as a seed-borne organism in temperate areas. l5 C. pallescens has been reported to occur on surface disinfected rice seeds and seeds of Brassica rapa and B.
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